Week 5: Friday

Question 1

Typical ER signal sequence

Answer 1

Signal sequences typically include 6-15 hydrophobic residues preceded by an arginine (R) or lysine (K). Often near the N-terminus

Question 2

Purpose of the positively charged amino acid in an ER signal sequence

Answer 2

The positively charged amino acid keeps the N-terminus in the cytosol

Question 3

What removes the signal peptide for many proteins that are targeted into the ER?

Answer 3

The signal peptide is removed by signal peptidase (a proteolytic enzyme).

Question 4

How are signal sequences inserted into the ER?

Answer 4

These signal sequences are inserted into the ER in a hairpin configuration (N-terminus stays on cytosolic side of the membrane)

Question 5

When polysomes translate a cytosolic protein, where is the mRNA?

Answer 5

The mRNA encoding a cytosolic protein remains free in cytosol.

Question 6

When polysomes translate a secretory protein with an N-terminal signal sequence, where is the mRNA?

Answer 6

mRNA encoding a protein targeted to the ER remains membrane-bound.

Question 7

What three things does the signal sequence direct to the ER membrane?

Answer 7

1. The ribosome
2. mRNA
3. The associated nascent chain

Question 8

The translocon

Answer 8

A pore in the ER through which the signal sequence directs the growing polypeptide chain.

Question 9

Co-translational translocation

Answer 9

The process by which a signal sequence directs the growing polypeptide chain into the lumen of the ER through the translocon. The signal sequence is then cleaved by signal peptidase in the lumen, making it a soluble component of the ER lumen.

Question 10

SRP

Answer 10

Signal recognition particle (the signal sequence receptor)

Question 11

What type of experiments were used to discover SRP?

Answer 11

Biochemical reconstitution experiments. They aim to test if a labeled protein was translocated into an organelle.

Question 12

Composition of SRP

Answer 12

SRP is composed of an RNA molecule and 6 protein subunits.

Question 13

Three steps of SRP’s function.

Answer 13

1. Recognizes signal sequence as it emerges from ribosome.
   2 .Causes a pause in translation.
2. Delivers ribosome with associated mRNA and partially translated protein to SRP receptor on ER membrane.

Question 14

Function of SRP receptor

Answer 14

The SRP receptor serves as a docking platform and it facilitates handoff of signal sequence to the translocon.

Question 15

What process controls the binding and release cycles of SRP?

Answer 15

The SRP receptor and SRP are both GTPases. The binding and release cycles are controlled by GTP hydrolysis.

Question 16

How was the translocon discovered?

Answer 16

The translocon was discovered by a yeast genetic approach.

Question 17

Why is a tight seal in the ER necessary during the translocation process?

Answer 17

The ER stores calcium, so a tight seal must be maintained during the process (Sec61 translocon) so it doesn’t escape.

Question 18

State of translocon when not engaged

Answer 18

A central plug closes the translocon when it isn’t engaged

Question 19

What happens to the translocon when the signal sequences binds to the receptor?

Answer 19

Binding of the signal sequence causes the plug to swing down and out of the channel. For many proteins, the signal sequence is cleaved off by the ER resident enzyme, signal peptidase.

Question 20

Which type of proteins require a cleavable signal sequence?

Answer 20

Soluble proteins that are secreted, or sorted to the lumen of organelles in the secretory or endocytic pathways, require a cleavable signal sequence.

Question 21

Structure of the translocon pore ring

Answer 21

The pore ring is made of the side chains of hydrophobic amino acids (mostly isoleucine). These side chains form a gasket around the polypeptide chain and brush away water and ions.

Question 22

What are the three modes of protein translocation?

Answer 22

1. Co-translational translocation (occurs in bacteria, archaea, and eukaryotes)
2. Post-translational translocation (eukaryotes)
3. Post-translational translocation (bacteria)

Question 23

What is mutated in patients with autosomal-dominant polycystic liver disease?

Answer 23

Sec63

Question 24

BiP is a member of what protein family?

Answer 24

BiP is a member of the Hsp70 family of ATPases that is localized to the ER lumen.

Question 25

Interaction between Sec63 and BiP

Answer 25

Sec63 interacts with BiP to help recruit this Hsp70 protein to the translocating peptide.

Question 26

SecA

Answer 26

An Hsp70 member protein required for translocation across the bacterial plasma membrane.

Question 27

Steps to the reconstitution approach

Answer 27

1. Break apart the cell
2. Purify the components
3. Put back together and see if the processes can be restored.